The following information is provided for the purpose of making known information believed by the applicants to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the following information constitutes prior art against the present invention.
Ribosome-inactivating proteins (RIPs) are plant proteins that are capable of catalytically inactivating eukaryotic ribosomes and are consequently extremely potent inhibitors of eukaryotic protein synthesis. RIPs have been divided into two classes: type 1 and type 2 RIPs (see Barbieri and Stirpe (1982), Cancer Surveys, 1:489-520). Type 2 RIPs (or A-chains are covalently attached via a disulfide bond to lectin-like proteins (or B-chains) with an affinity for cell surface carbohydrates. The B-chain binds to the cell surface and facilitates subsequent cellular internalization of the RIP A-chain moiety, which results in rapid inactivation of protein synthesis and cell death. Type 2 RIPs are therefore extremely potent cytotoxins and animal poisons, the best studied example of which is ricin.
In contrast, type 1 RIPs characterized to date consist of a single polypeptide chain equivalent in activity to that of A-chain RIPs but lacking a covalently attached B-chain. Consequently, they are scarcely toxic to intact cells but retain their extreme potency against cell-free protein translation systems. Typical concentrations that inhibit cell-free protein translation by 50% (IC.sub.50) are 0.5-10 ng/ml (0.16-33 pM). Until the discoveries detailed below, type 1 RIPs were a remarkably homogeneous class of basic proteins with Mr values of about 30,000. There is significant amino acid sequence homology between members of both type 1 and type 2 RIPs, and with the bacterial Shiga and Shiga-like toxins which also have the same mechanism of action (Hovde et al. (1988), Proc. Natl. Acad. Sci. USA, 85:2568-2572).
Where the effect of RIPs on ribosomes has been examined, both type 1 and type 2 RIPs possess a unique and highly specific N-glycosidase activity which cleaves the glycosidic bond of adenine 4324 of the ribosomal 28S RNA (Endo in Immunotoxins (1988), (ed.) Frankel).
Interest in RIPs has primarily focused on their use in construction of therapeutic toxins targeted to specific cells such as tumor cells by attachment of a targeting polypeptide, most frequently a monoclonal antibody (Immunotoxins (1988), supra. This mimics the binding functionality of the B-chain of type 2 RIPs but replaces their non-specific action with a highly selective ligand. Another recent potential use is in HIV therapy (see U.S. Pat. No. 4,869,903).
Type 1 RIPs are found in a great variety of dicot and monocot plants and they may be ubiquitous. They are often abundant proteins in seeds, roots or latex for example. Their in vivo function is unclear but it has been proposed that they may be antiviral (Stevens et al. (1981), Experientia 37:257-259) or antifungal (Roberts and Seltrennikkoff (1986), Bioscience Reports, 6:19-29) agents.
To date, one article has discussed the presence of an inhibitor of animal cell-free protein synthesis in maize, as well as other cereal crops (Coleman, W. H. and Roberts W. R. (1982), Biochimica et Biophysica Acta, 696:239-244. The preparation of the maize inhibitor was via ammonium sulfate precipitation and phosphocellulose column chromatography. It is generally believed that the inhibitor isolated from maize was pure. The reported molecular weight of the inhibitor was 23 kiloDaltons (kD), with a reported IC.sub.50 of 50-100 ng/ml in an ascites cell-free protein synthesis assay.
However, while a maize-derived ribosome inhibitor, like other ribosome inhibitors would appear to be useful for construction of cytotoxic conjugates, no artisan to date has reported to have successfully used a maize RIP. This is somewhat surprising in view of the success achieved with RIPs from other plants, including cereals such as barley (Lambert et al. in Immunotoxins, (1988), supra).
There is interest for recombinantly expressing RIP in commonly employed host eukaryotic cells. However, as RIPs effectively inhibit protein synthesis in eukaryotic cells, a predictable problem is that heterologous expression of an RIP will result in host cell death. Thus, eukaryotic cells are generally not used as recombinant host cells. Although eukaryotic cells may be used in certain circumstances, the RIP must be constructed so as to be secreted prior to the cell experiencing toxicity (see EP 0 335 476 A2). Therefore, prokaryotic host cells are the preferred hosts. Prokaryotic host cells, however, have disadvantages such as the inability to glycosylate and properly fold heterologously expressed proteins.
It is thus an object of the invention to provide a method of preparing inactive forms of RIPs, in which an inactive RIP may be expressed by eukaryotic host cells and then converted to an active form.
It is yet another object of the invention to provide the DNA sequence of the gene encoding such inactive forms of RIP, as well as plasmids host cells and cell cultures containing such DNA sequence.
Other objects and advantages of the present invention will become apparent from the Detailed Description of the Invention presented hereunder.
It is to these objects to which the present invention is directed.